Project Summary Innate immune responses involve the detection of foreign nucleic acids, including those from viruses. Multiple protein sensors detect the presence of viral RNA and DNA, leading to type I interferon (IFN) signaling and translational suppression of capped RNAs. Nevertheless, pathogens thwart this response by various mechanisms. Our published results revealed that unintegrated DNA containing specific retroviral sequences leads to increased cap-dependent translation of co-introduced genes (termed superinduction). High levels of lentiviral, gammaretroviral, and betaretroviral DNA resulted in superinduction in rat, mouse, and human cell lines using multiple transfection methods. Surprisingly, no differences were observed in type I IFN or mTOR signaling as well as the stress, DNA damage, or unfolded protein responses in the presence of retroviral sequences relative to standard plasmid vectors. These results led to the hypothesis that high levels of unintegrated retroviral DNA are detected by a factor that signals to the cap-binding complex to increase translation of capped RNAs from newly introduced DNAs. In the first specific aim, different parts of the superinduction pathway will be identified. An antibody-based screen that detects differences in phosphorylation in multiple signal transduction pathways will be used to detect signaling differences during superinduction. Protein-protein interactions with the translation initiation factor eIF-4E, which increases during superinduction, or other translation-related factors detected in the screen will be used to covalently trap and identify other signaling partners using a newly developed method, Ubiquitin-Activated Interaction Traps or UBAITs. Trapped proteins then will be purified and identified by mass spectroscopy. In the second specific aim, optimal sequences for superinduction will be defined by testing both engineered and synthesized retroviral and non-retroviral sequences after transduction or transfection. Using an optimized superinducing sequence compared to a non-inducing sequence, biotinylated DNAs will be introduced into cells, and the associated proteins will be purified using streptavidin beads and identified by mass spectrometry. The goal of these studies is to understand how mammalian cells respond to the presence of foreign DNA. This proposal has immediate short-term benefits for optimization of DNA transfection for protein expression as well as for retroviral transduction experiments. Long-term benefits include identification of the signaling pathways for detection of foreign nucleic acids as well as therapeutic targets with applications for gene therapy and anti-viral drugs.